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Biomedical subjects

F Pan

Publications and source records attributed to F Pan.

At least 73 records · Page 4Linked to original sources
PubMed1991

Reliability of stimulated and spontaneous growth hormone (GH) levels for identifying the child with low GH secretion.

The reliability of stimulated and spontaneous GH levels for identifying the child with low GH secretion has been the subject of debate. We compared the ability of GH concentrations after pharmacological stimulation with levodopa and clonidine and of spontaneous peak and 12-h pooled GH concentrations during sleep on a single night to estimate the maximum spontaneous GH secretion from 2 nights in 55 children, aged 5-16 yr, with heights below the 3rd percentile and/or height velocities below the 25th percentile for age, who had two consecutive overnight GH secretory profiles. Maximum stimulated GH concentrations correctly categorized 80% of children who had maximum spontaneous GH concentrations above and below 4 micrograms/L using a double monoclonal immunoradiometric assay for GH (Tandem-R HGH, Hybritech). The remaining 20% of children had stimulated GH concentrations below but spontaneous GH concentrations above 4 micrograms/L. Using this cut-off, the maximum GH concentrations from the first and second nights correctly categorized 98% and 95% of the children, respectively. Night to night variation in GH secretion was low in children who had low spontaneous GH secretion (maximum spontaneous peak and pool GH concentrations, less than 4 and less than or equal to 0.7 micrograms/L, respectively), and pooled GH concentrations from the 2 nights were concordant in 98% of the cases. We conclude that it is not uncommon for stimulated GH concentrations to underestimate spontaneous GH secretion. Even without acclimatization to the hospital setting, measurement of spontaneous GH secretion on a single night was more reliable for identifying the child with low endogenous GH secretion than was GH stimulation testing alone.

Adolescent↗
PubMed1985

Kinetic mechanism of glutaminyl-tRNA synthetase from human placenta.

The order of interaction of substrates and products with human placental glutaminyl-tRNA synthetase was investigated in the aminoacylation reaction by using the steady-state kinetic methods. The initial velocity patterns obtained from both the glutamine-ATP and glutamine-tRNA substrate pairs were intersecting, whereas ATP and tRNA showed double competitive substrate inhibition. Dead-end inhibition studies with an ATP analog, tripolyphosphate, showed uncompetitive inhibition when tRNA was the variable substrate. The product inhibition studies revealed that PPi was an uncompetitive inhibitor with respect to tRNA. The noncompetitive inhibition by AMP versus tRNA was converted to uncompetitive by increasing the concentration of glutamine from 0.05 to 0.5 mM. These and other kinetic patterns obtained from the present study, together with our earlier finding that this human enzyme catalyzed the ATP-PPi exchange reaction in the absence of tRNA, enable us to propose a unique two-step, partially ordered sequential mechanism, with tRNA as the leading substrate, followed by random addition of ATP and glutamine. The products may be released in the following order: AMP, PPi and then glutaminyl-tRNA. The proposed mechanism involves both a quarternary complex including all three substrates and the intermediary formation of an enzyme-bound aminoacyl adenylate, common to the usual sequential and ping-pong mechanisms, respectively, for other aminoacyl-tRNA synthetases.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Continuous spectrophotometric assay for aminoacyl-tRNA synthetases.

A simple, continuous assay for aminoacyl-tRNA synthetases utilizing a commercially available pyrophosphate assay reagent kit was demonstrated. The method coupled aminoacyl-tRNA synthetase activity with pyrophosphate-dependent fructose-6-phosphate kinase, aldolase, triosephosphate isomerase, and glycerophosphate dehydrogenase. PPi formation was correlated with the oxidation of NADH, and was monitored continuously by the decrease of absorbance at 340 nm.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Kinetic mechanism of arginyl-tRNA synthetase from human placenta.

Like arginyl-tRNA synthetases from other organisms, human placental arginyl-tRNA synthetase catalyzes the arginine-dependent ATP-PPi exchange reaction only in the presence of tRNA. We have investigated the order of substrate addition and product release of this human enzyme in the tRNA aminoacylation reaction by using initial velocity experiments and dead-end product inhibition studies. The kinetic patterns obtained are consistent with a random Ter Ter sequential mechanism, instead of the common Bi Uni Uni Bi ping-pong mechanism for all other human aminoacyl-tRNA synthetases so far investigated in this respect.

Adenosine Triphosphate↗
PubMed1983

Colorimetric assay for aminoacyl-tRNA synthetases.

A simple, rapid, and inexpensive procedure for the determination of aminoacyl-tRNA synthetase activity is described. The assay is based on a green color formed between PPi and ammonium molybdate in the presence of mercaptoethanol. The sensitivity is in the nanomole range and is comparable with the conventional [32P]PPi-ATP exchange assay. Amino acid, ATP, magnesium ion, and most common reagents do not interfere with the color yield.

Amino Acyl-tRNA Synthetases↗
PubMed1982

Kinetic mechanism of threonyl-tRNA synthetase from human placenta.

Using purified enzyme and homologous tRNA, we have investigated the order of substrate binding and product release for the human placental threonyl-tRNA synthetase by isotope exchange, initial velocity, dead-end inhibition and product inhibition studies. The kinetic patterns obtained from these studies are consistent with a unique Bi Uni Uni Bi ping-pong mechanism. The order of addition of the first two substrates ATP and threonine is random, while the release of products follows an obligatory sequence, with AMP as the last product to dissociate from the enzyme.

Adenosine Triphosphate↗
PubMed1982

Purification and subunit structure studies of human placental threonyl-tRNA synthetase.

Human threonyl-tRNA synthetase has been purified from full-term placenta over 5000-fold with a 13% overall yield by a combination of affinity chromatographic methods and conventional procedures. The product was apparently homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the esterification of approximately 3500 nmol L-threonine to tRNA per min per mg of protein at 37 degrees, corresponding to a molecular activity of approximately 700/min. The apparent molecular weight of the native enzyme ranged from 210000 to 220000 by gel-filtration on Sephadex G-200, and was either approximately 110000 or 228000 by sucrose gradient centrifugation for different preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a single band of molecular weight 85000 or 115000 for different preparations. These results suggest that human placental threonyl-tRNA synthetase has a subunit structure of the type alpha 2 with a subunit molecular weight of about 100000. However, some other molecular forms with lower specific activity were also found to exist under certain conditions.

Amino Acyl-tRNA Synthetases↗
PubMed1981

Periodate-oxidized AMP as a substrate, an inhibitor and an affinity label of human placental alkaline phosphatase.

Human placental alkaline phosphatase (EC 3.1.3.1) was inactivated by periodate-oxidized AMP. The inactivation showed saturation kinetics and could be partially prevented by the substrate AMP or the product inhibitor inorganic phosphate. Oxidized AMP was itself a substrate for this enzyme, with an apparent Km of 0.67 mM. The hydrolytic products of oxidized AMP were identified as oxidized adenosine hemiacetals. Oxidized AMP was also found to be a non-competitive inhibitor with respect to p-nitrophenyl phosphate, with identical Kis and Kii values of 0.15 mM. Our results indicate that oxidized AMP could combine with the enzyme to form a binary complex, followed by reaction with the proximal lysyl amino group to yield a Schiff base. The latter was reduced with NaBH4 and identified by t.l.c. The incorporation of only 1.5 molecules of oxidized [14C]AMP per enzyme subunit resulted in a complete inactivation of the enzyme. The modified enzyme showed higher apparent Km for the substrates and higher Ki for inorganic phosphate, but lower [32P]phosphate incorporation, than the native enzyme. These results support the conclusion that a lysine residue is involved in the phosphate-binding site of human placental alkaline phosphatase.

Adenosine Monophosphate↗
PubMed1979

Kinetic studies on human cysteinyl-tRNA synthetase.

The detailed pH and temperature kinetics of human term placenta cysteinyl-tRNA synthetase (EC 6.1.1.16) were studied. The ATP-PPi exchange reaction catalyzed by the cysteinyl-tRNA synthetase was highly dependent on temperature, pH, and ionic strength. The Arrhenius plot at temperatures between 5 degrees and 40 degrees was linear, giving an activation energy of 19 +/- 2.5 Kcal/mol. The pH dependence of the kinetic parameters Km and Vmax was investigated. Apparent pKa value of 6.4 was observed in the pH-dependence of Vmax/Km plot. The pH versus Vmax plot showed two apparent pKa values of about 5.8 and 7.8. Van't Hoff's enthalpies were used to differentiate the nature of the possible groups responsible for the ionization. These results are valuable for the selection of chemical modifying reagents in characterizing the amino acid residues involved in substrate (nucleotide) binding or catalysis.

Amino Acyl-tRNA Synthetases↗
PubMed1976

Multiple molecular forms of cysteinyl-tRNA synthetase from rat liver: purification and subunit structure.

Cysteinyl-tRNA synthetase (L-cysteine:tRNACys ligase (AMP-forming), EC 6.1.1.16) has been purified from rat liver in 23% overall yield. The enzyme was resolved by hydroxyapatite chromatography into three active forms (Fractions CRS-1, CRS-2 and CRS-3). The total activity ratio was about 0.7:2:1. The fractions CRS-2 and CRS-3 contained no other detectable aminoacyl-tRNA synthetase activity. CRS-2 was homogeneous by polyacrylamide gel electrophoresis, CRS-3 gave two active bands with mobilities corresponding to those of CRS-1 and CRS-2. The molecular weight of CRS-2 was about 240 000 by electrophoretic mobilities on the gels of various porosity, and 115 000-140 000 by sucrose gradient centrifugation. By gel-filtration, CRS-1, CRS-2 and CRS-3 exhibited apparent molecular weights of 122 000, 235 000 and 300 000, respectively. By sodium dodecyl sulfate gel electrophoresis, both CRS-2 and CRS-3 gave a single major band of 120 000 daltons. Stoichiometric study of cysteinyl adenylate formation indicated that CRS-2 has two active sites per molecule. These results are consistent with a dimeric structure of the type alpha2 for the major form of rat liver cysteinyl-tRNA synthetase, composed of two probably identical subunits of about 120 000 daltons. Available evidence also suggests that CRS-1 and CRS-3 are alpha and alpha3 (or alpha4), respectively.

Amino Acyl-tRNA Synthetases↗